PathHunter -Arrestin GPCR Assays - DiscoverX · 2 contents legal section intended use technology...

Contact Information

DiscoveRx Corporation

(World Wide Headquarters)

42501 Albrae Street

Fremont, CA 94538 United States

t | 1.510.979.1415

f | 1.510.979.1650

toll-free | 1.866.448.4864

DiscoveRx Corporation Ltd.

(Europe Headquarters)

Faraday Wharf, Holt Street Birmingham Science Park Aston

Birmingham, B7 4BB

United Kingdom

t | +44.121.260.6142

f | +44.121.260.6143

KINOMEscan® A division of DiscoveRx

11180 Roselle Street, Suite D

San Diego, CA 92121

United States

t | 1.800.644.5687

f | 1.858.630.4600

BioSeek® A division of DiscoveRx

310 Utah Avenue, Suite 100

South San Francisco, CA 94080

United States

t | 1.650.416.7600

f | 1.650.416.7625

www.discoverx.com

© 2014 DiscoveRx Corporation, Fremont, CA 94538. All rights reserved.

70-247 DRx_UM_PH_ARR_0914V7 Contact Information

Simple Solutions for Complex Biology

User Manual

PathHunter® β-Arrestin GPCR Assays For Chemiluminescent Detection of Activated GPCRs

2

CONTENTS

LEGAL SECTION INTENDED USE

TECHNOLOGY PRINCIPLE

ASSAY OVERVIEW

RECOMMENDED MATERIALS MATERIALS PROVIDED

ADDITIONAL MATERIALS REQUIRED (NOT PROVIDED)

FROZEN CELL HANDLING PROCEDURE

CELL PLATING REAGENT REQUIREMENTS

SOLVENTS AND PREPARATION OF COMPOUND DILUTIONS

USE OF PLASMA OR SERUM CONTAINING SAMPLES

STORING & REMOVING CRYOVIALS FROM LIQUID NITROGEN

CELL THAWING AND PROPAGATION

CELL FREEZING PROTOCOL

PREPARATION OF ASSAY PLATES

ASSAY PROCEDURE — AGONIST DOSE RESPONSE

PROTOCOL

QUICK START PROCEDURE

ASSAY PROCEDURE — ANTAGONIST DOSE RESPONSE

PROTOCOL


ASSAY PROCEDURE — ALLOSTERIC MODULATOR RESPONSE

PROTOCOL


ASSAY PROCEDURE — NEUTRALIZING ANTIBODY RESPONSE

PROTOCOL


ASSAY PROCEDURE — Ca2+ MOBILIZATION DOSE RESPONSE (Gq COUPLED RECEPTORS)

PROTOCOL


TROUBLESHOOTING GUIDE

APPENDIX A: ASSAY FORMATS

APPENDIX B: RELATED PRODUCTS

PAGE 3

PAGE 4

PAGE 4

PAGE 5

PAGE 5 PAGE 5

PAGE 6

PAGE 6

PAGE 6

PAGE 7

PAGE 7

PAGE 7

PAGE 8

PAGE 9

PAGE 10

PAGE 13 PAGE 14 PAGE 17

PAGE 18 PAGE 21

PAGE 22 PAGE 25

PAGE 26 PAGE 30

PAGE 31

PAGE 33

PAGE 33

35

NOTES:

34

NOTES:

3

LEGAL SECTION This product and/or its use is covered by one or more of the following U.S. patents #6,342,345 B1, #7,135,325 B2, #8,101,373 B2 and/or foreign patents, patent applications, and trade secrets that are either owned by or licensed to DiscoveRx® Corporation. This product is for in vitro use only and in no event can this product be used in whole animals. The right to use or practice the inventions in the foregoing patents (including method of use claims) by using or propagating this product is granted solely in connection with the use of appropriate Detection Reagents (protected under trade secret) purchased from DiscoveRx® Corporation or its authorized distributors.

LIMITED USE LICENSE AGREEMENT The cells and detection reagents (collectively Materials) purchased from DiscoveRx® are expressly restricted in their use. DiscoveRx has developed a Protein:Protein Interaction assay (Assay) that employs genetically modified cells and vectors (collectively, the ―Cells‖), and related detection reagents (the ―Reagents‖) (collectively referred to as ―Materials‖). By purchasing and using the Materials, the Purchaser agrees to comply with the following terms and conditions of this label license and recognizes and agrees to such restrictions:

1. The Materials are not transferable and will be used only at the site for which they were purchased. Transfer to another site owned by Purchaser will be permitted only upon written request by Purchaser followed by subsequent written approval by DiscoveRx.

2. The Reagents contain or are based upon the proprietary and valuable know-how

developed by DiscoveRx, and the Reagents have been optimized by DiscoveRx to function more effectively with the Cells in performing the Assay. Purchaser will not analyze or reverse engineer the Materials nor have them analyzed on Purchaser’s behalf.

3. In performing the Assay, Purchaser will use only Reagents supplied by DiscoveRx or an authorized DiscoveRx distributor for the Materials.

If the purchaser is not willing to accept the limitations of this limited use statement and/or has any further questions regarding the rights conferred with purchase of the Materials, please contact:

Licensing Department DiscoveRx Corporation 42501 Albrae Street

Fremont, CA 94538 USA tel | 1.510.979.1415 x104

[email protected] For some products/cell lines, certain 3rd party gene specific patents may be required to use the cell line. It is the purchaser's responsibility to determine if such patents or other intellectual property rights are required.

4

INTENDED USE

PathHunter® β-Arrestin GPCR Assays are whole cell, functional assays that directly

measure GPCR activity by detecting the interaction of β-Arrestin with the activated

GPCR. Because Arrestin recruitment occurs independent of G-protein coupling,

PathHunter β-Arrestin assays offer a powerful and universal screening platform that

can be used with virtually any Gi-, Gs-, or Gq-coupled receptor. This PathHunter system combines engineered clonal cell lines stably expressing the ProLink™ (PK)-

tagged GPCR of interest and the Enzyme acceptor (EA)-tagged β-Arrestin fusion

proteins with optimized PathHunter Detection Reagents (Cat. #93-0001, 93-0001L and 93-0001XL). Each cell line has been characterized for appropriate GPCR phar-macology, specificity and stability in cell culture. By combining a simple, one-step addition protocol and standard chemiluminescent detection, these assays are ideally suited for 96-well, 384-well, or 1536-well compound screening.

TECHNOLOGY PRINCIPLE

PathHunter β-Arrestin cell lines monitor GPCR activity by detecting the interaction

of β-Arrestin with the activated GPCR using β-galactosidase (β-gal) enzyme fragment

complementation (EFC, Figure 1). In this system, the GPCR of interest is fused

in frame with the small, 42 amino acid fragment of β-gal called ProLink™ and

co-expressed in cells stably expressing a fusion protein of β-Arrestin and the larger,

N-terminal deletion mutant of β-gal (called enzyme acceptor or EA). Activation of

the GPCR stimulates binding of β-Arrestin to the ProLink-tagged GPCR and forces

complementation of the two enzyme fragments, resulting in the formation of an

active β-gal enzyme. This action leads to an increase in enzyme activity that can

be measured using chemiluminescent PathHunter Detection Reagents. Because arrestin recruitment occurs independent of G-protein coupling, these assays provide a direct, universal platform for measuring receptor activation.

Figure 1. PathHunter β-Arrestin Assay Principle. Activation of the ProLink-tagged GPCR

results in β-Arrestin recruitment and formation of a functional enzyme capable of hydrolyzing

substrate and generating a chemiluminescent signal.

33

APPENDIX A: ASSAY FORMATS

*AssayComplete Cell Plating Reagent volume used to resuspend cells for assay plates

APPENDIX B: RELATED PRODUCTS

PathHunter® Certified Assay Format

Plate Format 96-well FV 384-well LV 384-well 1536-well

Total Volume 150 μL 40 μL 20 μL 8 μL

Cell Numbers 10,000 5,000 2,500 1,250

Cell Plating Reagents* 90 μL 20 μL 10 μL 4 μL

Ligand 10 μL 5 μL 2.5 μL 1 μL

Detection Reagents 50 μL 12 μL 6 μL 3 μL

Description Ordering Information

PathHunter® Detection Reagents www.discoverx.com/detectionreagents

Cell Culture Kits, Reagents & Consumables www.discoverx.com/cell-culture-kits-

reagents-consumables

AssayComplete™ Cell Plating Reagents www.discoverx.com/Cellplatingreagents

Control Ligands www.discoverx.com/controlligands

PathHunter® β-Arrestin Ortholog GPCR

Cell Lines

www.discoverx.com/GPCRtargets

PathHunter® eXpress β-Arrestin GPCR

Assays

www.discoverx.com/eXpressGPCRtargets

PathHunter® β-Arrestin Orphan GPCR

Cell Lines

www.discoverx.com/orphanGPCRtargets

32

PROBLEM CAUSE SOLUTION

Cells growing slowly U2OS grows slower than

CHO-K1 or HEK 293

Average doubling time is 3

days, so please observe cells

under microscope and

monitor cell health

Slow growing clones Use of DiscoveRx functional-

ly validated and optimized

media and reagents im-

proves assay performance

EC50 is right-shifted Improper ligand handling or

storage

Check ligand handling

requirements

Difference in agonist binding

affinity

Confirm that the ligand used

is comparable to the ligand

in the Product Insert

Problems with plate type and

compound stability

Hydrophobic compounds

should be tested for solubili-

ty and may be diluted in

buffer containing 0.1% BSA

Non-binding surface plates

may be necessary for

hydrophobic compounds

High well-to-well

variability in Z’ study

Problems with plate type and

compound solubility

Z’ studies should be

performed with automation

It may be necessary to test

plate types and compound

stability

TROUBLESHOOTING GUIDE (CONTINUED) For additional information or technical support, please call 1.866.448.4864 (US) +44.121.260.6142 (Europe) or email [email protected]

5

ASSAY OVERVIEW Please read the entire protocol completely before running the assay. The Assay Procedure sections and Quick Start Guides in this booklet contain detailed information about how to run the assays. Refer to the cell-line specific datasheet for additional information on the optimized AssayComplete™ Cell Plating Reagent and reference ligand recommended for the assay. Assays should be run using a fresh split of low-passage cells that have not been allowed to reach confluency for more than 24 hours. Following treatment of the cells with compound, GPCR activity is detected by adding a working solution of chemiluminescent PathHunter Detection Reagents using a simple, mix-and-read protocol. The following steps are required to monitor GPCR activity using a PathHunter

β-Arrestin GPCR cell line (Figure 2).

1. Plate cells (page 9).

2. Dilute and add compounds or antibodies.

3. Perform functional assay in agonist (page 10), antagonist (page 13) or allo-steric modulator mode (page 17).

Figure 2. Simple chemiluminescent assay protocol for monitoring GPCR activity in response to compound challenge.

MATERIALS PROVIDED

*Please refer to the cell line specific datasheet for detailed information on the PathHunter

β-Arrestin Cell Line you are testing.

RECOMMENDED MATERIALS The following materials are recommended:

CytoTracker™ Cell Proliferation Kit (DiscoveRx, Cat. # 92-2001M)

CytoTracker™ LDH Quantification Kit (DiscoveRx, Cat. # 92-2002)

CytoTracker™ Glutathione Quantification Kit (DiscoveRx, Cat. # 92-2003)

CytoTracker™ DNA Damage Quantification Kit (DiscoveRx, Cat. # 92-2004M)

Description Contents Storage

PathHunter β-Arrestin GPCR Cell Line 2 vials Liquid N2 (vapor phase)

Plate cells & add compounds

Add PathHunter Detection Reagents

Read Luminescence

6

ADDITIONAL MATERIALS REQUIRED (NOT PROVIDED)

The following additional materials are required to perform PathHunter β-Arrestin

GPCR Assays:

±Please refer to the cell line specific datasheet to determine catalog numbers for the media and

reagent requirements for the PathHunter β-Arrestin cell line you are testing.

FROZEN CELL HANDLING PROCEDURE To ensure maximum cell viability, thaw the vial and initiate the culture as soon as possible upon receipt. If continued storage of the frozen vials is necessary, store vials in the vapor phase of liquid nitrogen (N2). DO NOT store at –80°C for extended periods as this could result in significant loss in cell viability.

CELL PLATING REAGENT REQUIREMENTS

Each PathHunter β-Arrestin GPCR cell line has been validated for optimal assay

performance using the recommended AssayComplete Cell Plating (CP) Reagent and control ligand as indicated in the cell line specific datasheet. For optimal perfor-mance using this PathHunter Certified System, always use the Assay-Complete CP Reagent recommended for the cell line and DO NOT substitute at any time.

Equipment Materials

Green V-Bottom PP Ligand Dilution Plates,

10 plates/pack

(DiscoveRx, Cat. #92-0011)

96-well Clear Bottom TC treated, Sterile

WCB, FB w/lid, 10 plates/pack

(DiscoveRx, Cat. #92-0014) 384-well Clear Bottom TC treated, Sterile

WCB, FB w/lid, 10 plates/pack

(DiscoveRx, Cat. #92-0013) 384-well White Bottom TC treated, Sterile

w/lid, 10 plates/pack

(DiscoveRx, Cat. #92-0015) Disposable Reagent Reservoir

(Thermo Scientific, Cat. #8094 or similar)

Hemocytometer

Cryogenic Freezing Container

(Nalgene, Cat. #5100-0001 or similar)

Cryogenic Freezer Vials

(Fisher Scientific, Cat. #375418 or similar) Multimode or luminescence plate reader

Single and multi-channel pipettors and

pipette tips

Tissue culture disposables and plasticware

(T25 and T75 flasks, etc.)

PathHunter® Detection Kit

(DiscoveRx, Cat. #93-0001, #93-0001L

or #93-0001XL)

AssayComplete™ Revive Media

(DiscoveRx, Cat. #92-0016RM Series) AssayComplete™ Cell Culture Kit

(DiscoveRx, Cat. #92-

0018/19/20/21/22G Series) AssayComplete™ Preserve Freezing

Reagent (DiscoveRx, Cat. #92-0017FR

Series)

AssayComplete™Cell Detachment

Reagent (DiscoveRx, Cat. #92-0009)

AssayComplete™ Cell Plating Reagent

(DiscoveRx, Cat. #93-0563R Series) Phosphate buffered saline (PBS)

GPCR control agonist

GPCR test compound(s) and/or

antagonists

31

TROUBLESHOOTING GUIDE

PROBLEM CAUSE SOLUTION

No Response

Improper cell growth

conditions

See datasheet for cell culture

conditions

High DMSO/solvent

concentration

Maintain DMSO/solvent at

<1% in serial dilutions of

compounds.

Improper ligand used or

improper ligand incubation

time

See datasheet for recom-

mended ligand and assay

conditions

Improper preparation of

ligand (agonist or antagonist)

Refer to vendor specific

datasheet to ensure proper

handling, dilution and

storage of ligand

Improper time course for

induction

Optimize time course of

induction with agonist and

antagonist.

Decreased Response Higher passages give

reduced performance

PathHunter cells are stable

up to 10 passages. Use low

passage cells whenever

possible

Cells are not adherent and

exhibit incorrect morphology

Confirm adherence of cells

using microscopy

Low or No Signal Improper preparation of

detection reagents

Detection reagents should

be prepared just prior to use

and are sensitive to light.

Problem with cell growth,

cell viability, cell adherence

or cell density

See datasheet for cell

culture conditions.

Problem with microplate

reader

Microplate reader should be

in luminescence mode. Read

at 1 sec/well.

Experimental S:B does

not match datasheet

value

For cell pools, S:B may vary

greatly from passage to

passage or day to day

Prepare a clonal cell line or

use lower passage number

cells.

Repeat the assay

Confirm assay conditions

Improper preparation of

ligand (agonist or antagonist)

Some ligands are difficult

to handle. Confirm the final

concentration of ligands

30

QUICK-START PROCEDURE: Ca2+ MOBILIZATION DOSE RESPONSE AGONIST ANTAGONIST

Plate 20 µL PathHunter cells per well

Incubate

overnight @ 37°C

Aspirate media and add 20 μL

Calcium NoWashPLUS working reagent

Incubate

60 minutes

@ 37°C

Add 10 µL

of buffer


Incubate

overnight @ 37°C

Aspirate media and add 20 μL

Calcium NoWashPLUS working reagent

Incubate

60 minutes

@ 37°C

Scan fluorescent

signal over 2 minutes

Add 10 μL

of 3X antagonist in buffer

Equilibrate for 30

minutes at room

temperature

Add 10 μL

of 4X EC80 Agonist in buffer

Equilibrate for 30

minutes at room

temperature

Scan fluorescent

signal over 2 minutes

Add 10 μL

of 4X Agonist in buffer

7

SOLVENTS AND PREPARATION OF COMPOUND DILUTIONS

PathHunter β-Arrestin GPCR assays are routinely carried out in the presence of ≤ 1%

solvent (i.e. DMSO, ethanol, PBS or other). As solvents can affect assay performance, optimize the assay conditions accordingly if other solvents or solvent concentrations are required.

To validate each PathHunter β-Arrestin GPCR Assay, reference ligand was diluted

using the AssayComplete Cell Plating (CP) Reagent recommended for the cell line (containing the appropriate solvent). For antibodies or other compounds that may be sensitive to serum and/or other assay components, dilutions can be prepared in either Hanks Buffered Salt Solution (HBSS) + 10 mM HEPES + 0.1% Bovine Serum Albumin (BSA) or OptiMEM® + 0.1% BSA without affecting assay performance.

USE OF PLASMA OR SERUM CONTAINING SAMPLES

PathHunter β-Arrestin GPCR Assays can be run in the presence of high levels of serum

or plasma without negatively impacting assay performance. Standard curves of control ligand can be prepared in neat, heparinized plasma and added directly to the cells (without further dilution, ie. 100% plasma in the well). After ligand stimulation, the samples should be removed and replaced with fresh AssayComplete CP Reagent before the addition of the PathHunter Detection Reagents. Refer to page 21 for more information.

NOTE:

EDTA anti-coagulated plasma samples do not give a positive response in the assay. Therefore,

the choice of anti-coagulant treatment is very important.

STORING & REMOVING CRYOVIALS FROM LIQUID NITROGEN Cells are shipped in 2 vials on dry ice and contain approximately 1 x 106 cells per vial in 1 mL of AssayComplete Preserve Freezing Reagent. The following proce-dures are for safely storing and removing cryovials from liquid nitrogen storage.

1. PathHunter cells must arrive in a frozen state on dry ice. If cells arrive thawed, do not proceed, contact technical support.

2. Frozen cells must be immediately transferred to liquid N2 storage or thawed and put into culture upon arrival.

3. When removing cryovials from liquid N2 storage, use tongs and place immediately on dry ice in a covered container. Wait at least one minute for any liquid N2 inside the vial to evaporate.

4. Proceed with the thawing protocol in the following section. SAFETY WARNING: A face shield, gloves and lab coat should be worn at all times when handling frozen vials. Some cryovials can leak when submerged in liquid N2. Upon thawing, the liquid N2 present in the cryovial converts back to its gas phase which can result in the vessel exploding.

8

CELL THAWING AND PROPAGATION The following procedures are for thawing, seeding and expanding the cells, and for maintaining the cultures once the cells have been expanded. Cells are free of contamination prior to shipment and care should be taken in their handling to avoid contamination.

NOTE:

Face shield, gloves and a lab coat should be worn during the thawing procedure.

1. Pre-warm 15 mL AssayComplete Revive Media in a 37°C water bath.

2. Place the frozen cell vials briefly (10 seconds to 1 min) in a 37°C water bath under sterile conditions until only small ice crystals remain and the cell pellet is almost completely thawed. Caution: Longer incubation may result in cell death.

3. To remove DMSO from the media, carefully transfer the thawed cells to a sterile 15 mL tube and then fill tube with 10 mL pre-warmed AssayComplete Revive Media. Centrifuge at 300 x g for 4 minutes to pellet cells.

4. Remove media without disturbing cell pellet and resuspend in 5 mL of pre-warmed AssayComplete Revive Media. Transfer cells to a T25 flask and incu-bate for 24 hours at 37°C, 5% CO2.

NOTE:

Cell recovery is greatly improved when selection antibiotics are omitted for the

first 24 hours.

5. After 24 hours, gently remove AssayComplete Revive Media (being careful not

to disturb the cell monolayer) and replace with 5 mL of pre-warmed complete AssayComplete Cell Culture Media.

6. Once the cells become >70% confluent in the T25 flask, aspirate media and wash cells with 5 mL PBS. Aspirate PBS and dissociate cells with 0.5 mL AssayComplete Cell Detachment Reagent and resuspend in 5 mL of Assay-Complete Cell Culture Media. Transfer the entire cell suspension to a T75 flask containing 15 mL of AssayComplete Cell Culture Media for continued growth.

7. Passage the cells every 2-3 days, based on the doubling time of the cell line, using AssayComplete Cell Detachment Reagent. For routine passaging, pre-pare a 1:3 dilution of cells in a total volume of 15 mL AssayComplete Cell Cul-ture Media. Transfer 5 mL of the diluted cells to each new T75 flask.

NOTE: To maintain logarithmic growth of the cells, cultures should be maintained in a

subconfluent monolayer.

8. Each PathHunter β-Arrestin GPCR Cell Line has been found to be stable for at

least 10 passages with no significant drop in assay window or shift in EC50.

9. Assay performance and cellular response can be assessed by treating the cells with reference agonist. Refer to the cell line specific datasheet for the

recommended control agonist for your PathHunter β-Arrestin GPCR

Cell Line. For antagonist assays, cells can be pretreated with varying doses of antagonist/inhibitor compounds followed by agonist challenge, typically at an EC80 concentration.

29

REPRESENTATIVE DATA Figure 10. PathHunter® CHO-K1 BDKRB2 β-Arrestin Cells (93-0189C2). Cells were

plated in a 384-well plate at 10,000 cells/well. Target cells and native parental cells were then

stimulated with the known agonist Bradykinin (DiscoveRx, Cat. # 92-1053). Calcium mobiliza-

tion was detected using the Calcium NoWashPLUS Detection Kit according to the recommended

protocol. The EC50 for agonist was estimated at 0.17 nM.

28

DAY 2: AGONIST COMPOUND ADDITION AND SIGNAL DETECTION

1. Aspirate media from cells and replace with 20 µL Ca NWPLUS working reagent.

2. Incubate cells for 1 hour at 37°C, 5% CO2.

3. Add 10 µL of HBSS/20 mM HEPES and equilibrate assay plate for 30 minutes

at room temperature.

4. Prepare serial dilutions of the compound for a 12-point dose response curve. We recommend that the highest final concentration be 50X the expected EC50 of the compound.

5. Add 10 µL of 4X agonist in HBSS/20 mM HEPES.

6. Measure compound activity using fluorescence reader with appropriate settings (excitation at 494 nM and emission at 516 nM). Signal is monitored over 2 minutes at 2 second intervals. We recommend that you pre-read the assay plates for 5 seconds prior to agonist addition.

ANTAGONIST COMPOUND ADDITION AND SIGNAL DETECTION

1. Aspirate media from cells and replace with 20 µL Ca NWPLUS working reagent.

2. Incubate cells for 1 hour at 37°C, 5% CO2.

3. Prepare serial dilutions of the compound for a 12-point dose response curve. We recommend that the highest final concentration be 50X the expected IC50 for the compound.

4. Add 10 µL of 3X antagonist in HBSS/20 mM HEPES, and equilibrate assay

plate for 30 minutes at room temperature.

5. Add 10 µL 4X EC80 agonist in HBSS/20 mM HEPES.

6. Measure compound activity using fluorescence reader with appropriate settings (excitation at 494 nM and emission at 516 nM). Signal is monitored over 2 minutes at 2 second intervals. We recommend that you pre-read the assay plates for 5 seconds prior to agonist addition.

DATA ANALYSIS 1. Dose curves in the presence and absence of compound are plotted using

GraphPad Prism or Activity Base.

2. For agonist mode assays, percentage activity is calculated using the following formula:

% Activity = 100% x (Mean RLU of test sample - Mean RLU of vehicle control) / (Mean MAX RLU control ligand - Mean RLU of vehicle control)

3. For antagonist mode assays, percentage inhibition is calculated using the following formula:

% Inhibition = 100% x (1 - (Mean RLU of test sample - Mean RLU of vehicle control) / (Mean RLU of EC80 control - Mean RLU of vehicle control)

9

CELL FREEZING PROTOCOL The following procedures are for freezing cells from confluent T225 flasks. If smaller flasks are used, adjust the volumes accordingly. Care should be taken in handling to avoid contamination.

1. Remove T225 flasks from incubator and place in the tissue culture hood. Aspirate the media from the flasks.

2. Add 10 mL PBS into each T225 flask and swirl to rinse the cells. Aspirate PBS from flask.

3. Add 5 mL of AssayComplete Cell Detachment Reagent to the flask. Rock the flask back and forth gently to ensure the surface of the flask is covered. Incu-bate at 37°C, 5% CO2 for 2–5 minutes or until the cells have detached.

4. Remove the flask from the incubator and view under a microscope to confirm that the cells have detached. If necessary, tap the edge of the flask to detach cells from the surface.

5. Add 8–10 mL of AssayComplete Revive Media to each T225 flask. Rinse the cells from the surface of the flask using the added media. Remove the cells from the flask and transfer to a 50 mL conical tube. (If necessary, add an additional 5 mL of media to the flask and rinse to collect the remaining cells and transfer the additional volume to the 50 mL conical tube). Remove 0.5 mL of the resuspended cells and count the cells using a hemocytometer.

6. Centrifuge the collected cells at 300 x g for 4 minutes.

7. After centrifugation, discard the supernatant. Resuspend the cell pellet in AssayComplete Preserve Freezing Reagent. Based on the cell number ob-tained from Step 5, dilute the resuspended cells to a concentration of 1.2 x 106 cells/mL using AssayComplete Preserve Freezing Reagent.

8. Transfer 1 mL cells to each 2 mL cryogenic tube. (Keep cells on ice during this process and transfer to a cryogenic container pre-chilled at 4°C).

9. Transfer tubes to –80°C and store overnight. Transfer tubes into the vapor phase of a liquid N2 tank for long-term storage.

PREPARATION OF ASSAY PLATES Each PathHunter β-Arrestin GPCR Assay has been validated for optimal assay per-formance using the specific AssayComplete Cell Plating Reagent. Always use the AssayComplete CP Reagent recommended for the cell line and DO NOT substitute at any time. 1. Harvest the cells as follows from a confluent T25 or T75 flask using Assay-

Complete Cell Detachment Reagent. Do not use Trypsin.

a) Remove AssayComplete Cell Culture Media.

b) Gently wash cells with 5 mL PBS and aspirate.

c) Add 0.5 mL AssayComplete Cell Detachment Reagent to each T25 flask, or 1 mL to each T75 flask.

d) Place the flask in the incubator for 5 minutes or until cells have detached.

e) Add 3 mL of CP Reagent and transfer to a 15 mL conical tube.

10

2. Determine the cell density using a hemocytometer. Centrifuge the cells at 300 x g for 4 minutes to pellet cells. Remove supernatant.

3. Resuspend cells in AssayComplete CP Reagent at a concentration of 250,000

cells/mL (5,000 cells/20 μL). Transfer 20 μL of the cell suspension to each

well of a 384-well microplate. Please refer to Appendix A for cell numbers and volumes for alternate formats.

4. Incubate the plate overnight at 37°C, 5% CO2.

ASSAY PROCEDURE — AGONIST DOSE RESPONSE CURVE The steps outlined below provide the assay volumes and procedures for performing

GPCR agonist assays using the PathHunter β-Arrestin GPCR Cell Lines and PathHunter

Detection Reagents in a 384-well format. Refer to Appendix A for cell numbers and volumes for alternate formats. Although plate layouts and experimental designs may vary, we recommend performing a 12-point dose curve for each compound using at least duplicate wells for each dilution.

DAY 1: PREPARATION OF ASSAY PLATES Plate PathHunter cells in the appropriate number of wells in a 384-well plate as described in the ―Preparation of Assay Plates‖ section on page 8. Allow cells to incu-bate overnight.

Figure 3. This plate map shows 12-point dose curves with 2 data points at each concentration.

Plate map allows 16 compounds to be tested in duplicate per 384-well plate.

3-fold serial

dilutions of agonist HighLow

1 2 3 4 5 6 7 8 9 10 11 12

A

BCompound 1 Compound 2

Compound 3 Compound 4

No a

gon

ist

C

D

E

F

G

H

I

J

K

LM

N

O

P

13 14 15 16 17 18 19 20 21 22 23 24







HighLow

No a

gon

ist

3-fold serial

dilutions of agonist

27

3. To prepare working reagent for one microplate, combine the following compo-nents and mix well:

a) 9 mL of Dye Loading Buffer.

b) 1 mL of Additive A. NOTE: Add Additive A before the dye.

c) 10 μL of reconstituted Ca NWPLUS Dye.

d) 100 μL of freshly prepared 250 mM probenecid solution.

4. Working reagent is stable for 24 hours at room temperature. Total signal may

drop over longer time periods. Prepare Ca NWPLUS working reagent as needed.

DAY 1: PREPARATION OF ASSAY PLATES Each PathHunter GPCR Assay has been validated for optimal assay performance using the specific AssayComplete Cell Plating (CP) Reagent. Always use the Assay-Complete CP Reagent recommended for the cell line and DO NOT substitute at any time. 1. Harvest the cells as follows from a confluent T25 or T75 flask using Assay-

Complete Cell Detachment Reagent. Do not use Trypsin.

a) Remove AssayComplete Cell Culture Media.

b) Gently wash cells with 5 mL PBS and aspirate.

c) Add 0.5 mL AssayComplete Cell Detachment Reagent to each T25 flask, or 1 mL to each T75 flask.

d) Place the flask in the incubator for 5 minutes or until cells have detached.

e) Add 3 mL of AssayComplete CP Reagent and transfer to a 15 mL conical tube.

2. Determine the cell density using a hemocytometer. Centrifuge the cells at 300 x g for 4 minutes to pellet cells. Remove supernatant.

3. Resuspend cells in AssayComplete CP Reagent at a concentration of 500,000

cells/mL (10,000 cells/20 μL). Transfer 20 μL of the cell suspension to each

well of a black 384-well microplate (DiscoveRx, Cat. # 92-0024). If using 96-

well plate seed 50,000 cells/100 μL in each well.

4. Incubate the plate overnight at 37°C, 5% CO2.

26

ASSAY PROCEDURE — Ca2+ MOBILIZATION DOSE RESPONSE (Gq COU-PLED RECEPTORS) The steps outlined below provide the assay volumes and procedures for performing

Ca2+ mobilization assays using the PathHunter β-Arrestin GPCR Cell Lines and Calcium

No WashPLUS (CaNWPLUS) Detection Kit (DiscoveRx, Cat. # 90-0091). Although plate layouts and experimental designs may vary, we recommend performing a 12-point dose curve for each compound using at least duplicate wells for each dilution. Sig-nal is measured on a fluorescent plate reader equipped with fluidic handling, capable of detecting rapid changes in fluorescence upon compound stimulation.

Figure 9. This plate map shows 12-point dose curves with 2 data points at each concentration. Plate map allows 16 compounds to be tested in duplicate per 384-well plate.

PREPARATION OF REAGENTS Preparation of Ca NWPlus Working Reagent: 1. Thaw reagents to room temperature and mix gently.

2. Add 110 μL anhydrous DMSO to 500 μg Ca NWPLUS Dye tube and vortex to

reconstitute dye. Allowing the pellet to soak in DMSO will facilitate reconstitution. It is very important to ensure that the dye goes completely into solution (solution should be dark yellow).

a) Store reconstituted dye at -20°C.

b) This reconstituted dye in DMSO reagent can be frozen and thawed up to 3 times without loss in performance.

c) Aliquot if necessary.

3-fold serial

dilutions of agonist HighLow

1 2 3 4 5 6 7 8 9 10 11 12

A

BCompound 1 Compound 2


No a

gon

ist

C

D

E

F

G

H

I

J

K

LM

N

O

P

13 14 15 16 17 18 19 20 21 22 23 24







HighLow

No a

gon

ist

3-fold serial

dilutions of agonist

11

DAY 2: AGONIST COMPOUND PREPARATION AND ADDITION 1. Dissolve agonist compound in the vehicle of choice (DMSO, ethanol, PBS or

other) at the desired stock concentration.

2. Prepare a series of twelve 3-fold serial dilutions of agonist compound in Assay-Complete CP Reagent containing the appropriate solvent (DMSO, ethanol, PBS or other) as described below. The concentration of each dilution should be

prepared at 5X of the final screening concentration (i.e. 5 µL compound + 20

µL of cells). For each dilution, the final concentration of solvent should remain

constant.

To prepare the 12-point dose curve serial dilutions, we recommend starting with a concentration that is 50X the expected EC50 value for the compound (e.g. 250X EC50 would be the final working concentration).

Example: If the expected EC50 is 10 nM, prepare the highest starting concen-

tration of the corresponding dilution at 2.5 µM. This is the working concentration.

a) For each compound tested, label the wells of a 384-well dilution plate #1 through #12.

b) Add 20 µL of AssayComplete CP Reagent containing appropriate solvent to

wells #1-11.

c) Prepare a working concentration of agonist compound in the appropriate AssayComplete CP Reagent.

d) Add 30 µL of the working concentration of agonist compound to well #12.

e) Remove 10 µL of compound from well #12, add it to well #11 and mix

gently by pipetting up and down. Discard pipet tip.

f) With a clean pipet tip, remove 10 µL of diluted compound from well #11,

add it to well #10 and mix gently by pipetting up and down. Discard the pipet tip.

g) Repeat this process 8 more times in succession to prepare serial dilutions for the remaining wells, from right to left across the plate.

DO NOT add agonist compound to well #1. This sample serves as the no agonist control and completes the dose curve.

h) Repeat this process for each additional agonist compound to be tested.

i) Set compounds aside until agonist compounds are ready to be added.

3. Remove PathHunter cells from the incubator (previously plated on day 1).

NOTE: 93-0203C7 PathHunter® C2C12 CXCR4 β-Arrestin Cell Line uses an additional media exchange step. Please refer cell line specific datasheet.

4. Transfer 5 µL from wells #1–12 to duplicate wells according to the plate map

shown on page 9.

5. Incubate for 90 minutes @ 37°C.

12

SUBSTRATE PREPARATION AND ADDITION 1. Prepare PathHunter Detection Reagent by combining 1 part Substrate Reagent

2 with 5 parts Substrate Reagent 1, and 19 parts of Cell Assay Buffer.

NOTE:

The working solution is stable for up to 8 hours at room temperature.

2. Add 12 μL of prepared detection reagent to the appropriate wells. DO NOT

pipet up and down in the well to mix or vortex/shake plates.

3. Incubate for 60 minutes at room temperature (23°C).

4. Read samples on any standard luminescence plate reader.

5. Use GraphPad Prism® or other comparable program to plot your agonist dose response. See the example shown in Figure 4.

REPRESENTATIVE DATA AND DATA ANALYSIS Figure 4. PathHunter® CHO-K1 SSTR2 β-Arrestin Cells (93-0181C2). Cells were plated

in a 384-well plate at 5,000 cells/well and stimulated with the known agonist Somatostatin 28

(DiscoveRx, Cat. # 92-1068) for 90 minutes. Signal was detected using the PathHunter Detec-

tion Kit (93-0001) according to the recommended protocol. An assay window of 52.4-fold S:B was

achieved in this example, and the EC50 for agonist was estimated at 1.8 nM.

Component Entire Plate (384 wells)

Cell Assay Buffer 4.75 mL

Substrate Reagent 1 1.25 mL


10 - 1 3 10 - 1 2 10 - 1 1 10 - 1 0 10 - 9 10 - 8 10 - 7 10 - 60

2500

5000

7500

10000

12500

15000

17500

20000

Somatostatin 28 [M]

RL

U

25

QUICK-START PROCEDURE: NEUTRALIZING ANTIBODY DOSE RESPONSE *Please refer to the cell line specific datasheet any variations in assay conditions.


Incubate

overnight

@ 37°C

Add 2.5 µL of Diluted Antibody

Incubate

30 minutes

@ 37°C

Read Chemiluminescent Signal

Add 12 µL

Detection Reagent Working Solution

Incubate

60 Minutes @

Room Temperature

Add 2.5 µL

of Agonist @ EC80

Incubate

90 minutes

@ 37°C*

Remove serum-containing sample.

Add 25 µL CP Reagent

FOR SERUM

SAMPLES ONLY!

24

ATTENTION! PLASMA OR SERUM-CONTAINING SAMPLES ONLY 5. After incubation is complete, gently aspirate the plasma or serum-containing

samples from the well. Be careful to remove as much sample as possible without disturbing the cell monolayer.

6. Immediately add 25 μL of fresh CP Reagent to each well. Proceed with substrate

preparation and addition.

SUBSTRATE PREPARATION AND ADDITION 1. Prepare PathHunter Detection Reagent by combining 1 part Substrate

Reagent 2 with 5 parts Substrate Reagent 1, and 19 parts of Cell Assay Buffer.

NOTE: The working solution is stable for up to 8 hours at room temperature.

2. Add 12 μL of prepared detection reagent to the appropriate wells and incubate

for 60 minutes at room temperature (23°C). DO NOT pipette up and down in the well to mix or vortex/shake plates.



5. Use GraphPad Prism® or other comparable program to plot your dose response.





13


Incubate

overnight

@ 37°C

Add 5 µL of Agonist

Incubate

90 minutes

@ 37°C*

Read

Chemiluminescent Signal

Add 12 µL


Incubate

60 Minutes

@ Room

Temperature

QUICK-START PROCEDURE: AGONIST DOSE RESPONSE *Please refer to the cell line specific datasheet for any variations in assay conditions.

14

ASSAY PROCEDURE — ANTAGONIST DOSE RESPONSE CURVE The steps outlined below provide the assay volumes and procedures for performing

GPCR antagonist assays using the PathHunter β-Arrestin GPCR Cell Lines and

PathHunter Detection Reagents in a 384-well format. Refer to Appendix A for cell numbers and volumes for alternate formats. Although plate layouts and experi-mental designs may vary, we recommend performing a 11-point dose curve for each compound using at least duplicate wells for each dilution. The protocol and volumes described below are designed for a complete 384-well plate.

Figure 5. This plate map shows 11-point dose curves with 2 data points at each concentration. Plate map allows 16 compounds to be tested in duplicate per 384-well plate.

DAY 1: PREPARATION OF ASSAY PLATES Plate PathHunter cells in the appropriate number of wells in a 384-well plate as described in the ―Preparation of Assay Plates‖ section on page 9. Allow cells to incubate overnight. DAY 2: ANTAGONIST COMPOUND PREPARATION AND ADDITION

1. Dissolve your antagonist compound in the vehicle of choice (DMSO, ethanol,

PBS or other) at the desired stock concentration.

2. Prepare a series of eleven 3-fold serial dilutions of antagonist compound in CP Reagent containing the appropriate solvent (DMSO, ethanol, PBS or other) as described below. The concentration of each dilution should be prepared at 10X

the final screening concentration (i.e. 2.5 µL antagonist compound will be used

in a final volume of 25 µL). For each dilution, the final concentration of solvent

23

To prepare the 11-point dose curve serial dilutions, we recommend starting with a concentration that is 50X the expected IC50 value for the compound (e.g. 500X IC50 would be the final working concentration).

Example: If the expected IC50 is 10 nM, prepare the highest starting concen-

tration of the corresponding dilution at 5 µM. This is the working concentration.

a) For each antibody tested, label the wells of a 384-well dilution plate #1 through #12.


wells #1-11.

c) Prepare a working concentration of antibody in the appropriate Assay-Complete CP Reagent.

d) Add 30 µL of the working concentration of antibody to well #12.

e) Remove 10 µL of antibody from well #12, add it to well #11 and mix gently

by pipetting up and down. Discard pipet tip.

f) With a clean pipet tip, remove 10 µL of diluted antibody from well #11,


g) Repeat this process 7 more times in succession to prepare serial dilutions for the remaining wells, from right to left across the plate. DO NOT add antibody to wells #1 and 2. This sample serves as the no antibody control and completes the dose curve.

h) Repeat this process for each additional antibody to be tested.

i) Set antibodies aside until they are ready to be added.


4. Transfer 2.5 µL from wells #1–12 to duplicate wells according to the plate map

shown on page 21.


AGONIST COMPOUND PREPARATION AND ADDITION 1. During the antibody incubation, determine the EC80 concentration of the agonist

to be used in the assay. Prepare a 10X EC80 concentration of agonist compound as shown below:

Example: If the expected EC80 of the agonist compound is 10 nM, prepare a stock at 100 nM.

2. Add 2.5 µL of agonist to each well. Add 2.5 µL of CP Reagent containing appro-

priate solvent to the no agonist wells (columns 1 & 13 in figure 8).


4. If samples do not contain plasma or serum, omit steps 5 and 6 and proceed

directly to the substrate preparation and addition.

22

ASSAY PROCEDURE — NEUTRALIZING ANTIBODY DOSE RESPONSE The steps outlined below provide the assay volumes and procedure for performing

detection of anti-GPCR neutralizing antibodies using the PathHunter β-Arrestin

GPCR Cell Lines and PathHunter Detection Reagents in a 384-well format. Refer to Appendix A for cell numbers and volumes for alternate formats. Although plate layouts and experimental designs may vary, we recommend performing an 11-point dose curve for each compound using at least duplicate wells for each dilution. The protocol and volumes described below are designed for a complete 384-well plate.

Figure 8. This plate map shows a 11-point dose curves with 2 data points at each concentration.

Plate layout allows 16 antibodies to be tested in duplicate per 384-well plate.

DAY 1: PREPARATION OF ASSAY PLATES Plate PathHunter cells in the appropriate number of wells in a 384-well plate as described in the ―Preparation of Assay Plates‖ section on page 9. Allow cells to incubate overnight.

DAY 2: ANTIBODY PREPARATION AND ADDITION 1. Dissolve antibody in the vehicle of choice (PBS, water or other) at the desired

stock concentration.

2. Prepare a series of eleven 3-fold serial dilutions of antibody in Cell Plating Reagent

containing the appropriate solvent (PBS, water or other). The concentration of each dilution should be prepared at 10X of the final screening concentration

(i.e. 2.5 µL antibody will be used in a final volume of 25 µL). For each dilution,

the final concentration of solvent should remain constant.

15

should remain constant. To prepare the 11-point dose curve serial dilutions, we recommend starting with a concentration that is 50X the expected IC50 value for the compound (e.g. 500X IC50 would be the final working concentration).





wells #1-11.

c) Prepare a working concentration of antagonist compound in the appropriate AssayComplete CP Reagent.

d) Add 30 µL of the working concentration of antagonist compound to well #12.


gently by pipetting up and down. Discard the pipet tip.



g) Repeat this process 7 more times in succession to prepare serial dilutions for the remaining wells, or from right to left across the plate. DO NOT add antagonist compound to tubes #1 and 2. These samples serve as the no antagonist controls and complete the dose curve.

h) Repeat process for any additional antagonist compounds to be tested.

i) Set compounds aside until you are ready to add them to the cells.



on page 13.

5. Incubate cells with antagonist compounds for 30 minutes @ 37°C.

AGONIST COMPOUND PREPARATION AND ADDITION 1. During the antagonist incubation, determine the EC80 concentration of the

agonist from the agonist dose response curve (described on pages 10-12). Prepare a 10X EC80 concentration of agonist compound in the appropriate CP Reagent/solvent as shown below:

Example: If the expected EC80 of the agonist compound is 10 nM, prepare a stock at 100 nM.

2. When the antagonist incubation is complete, add 2.5 µL of agonist compound

to wells #2–12. Add 2.5 µL of CP Reagent containing appropriate solvent to

the ―No antagonist/No agonist‖ wells (columns 1 & 13 in Figure 5).


16



NOTE:


2. Add 12 μL of prepared detection reagent to the appropriate wells. DO NOT

pipet up and down in the well to mix or vortex/shake plates.



5. Use GraphPad Prism® or other comparable program to plot your antagonist dose response.

REPRESENTATIVE DATA AND DATA ANALYSIS

Figure 6. PathHunter® CHO-K1 ADRB2 β-Arrestin Cells (93-0182C2). Cells were plated

in a 384-well plate at 5,000 cells/well and levels of β-Arrestin recruitment was measured after 30

minutes of pre-incubation with the indicated concentrations of antagonist compounds followed

by a 90 minute incubation with a single EC80 concentration of isoproterenol (DiscoveRx; 92-1119).

Signal was detected using the PathHunter Detection Kit (93-0001) according to the recommended

protocol.





10- 1 1 10- 1 0 10- 9 10- 8 10- 7 10- 6 10- 5 10- 4 10- 3

0

10000

20000

30000Propanolol

Timolol

CGP12177a

Carvedilol

Atenolol

Nadolol

Antagonist [M] + 500 nM Isoproterenol

RL

U

21


Incubate

overnight

@ 37°C

Add 2.5 µL of

Allosteric Modulator Compound

Incubate

30 minutes

@ 37°C

Read

Chemiluminescent Signal

Add 12 µL


Incubate

60 Minutes @

Room Temperature

Add 2.5 µL

of Agonist

Incubate

90 minutes

@ 37°C*

QUICK-START PROCEDURE: ALLOSTERIC MODULATOR DOSE RESPONSE *Please refer to the cell line specific datasheet for any variations in assay conditions.

20



NOTE:


2. Add 12 μL of prepared detection reagent to the appropriate wells and incubate

for 60 minutes at room temperature (23°C). DO NOT pipette up and down in the well to mix or vortex/shake plates.



5. Use GraphPad Prism® or other comparable program to plot your allosteric modulator dose response.





17

QUICK-START PROCEDURE: ANTAGONIST DOSE RESPONSE *Please refer to the cell line specific datasheet for any variations in assay conditions.


Incubate

overnight

@ 37°C

Add 2.5 µL

of Antagonist

Incubate

30 minutes

@ 37°C

Read Chemiluminescent Signal

Add 12 µL


Incubate

60 Minutes @

Room Temperature

Add 2.5 µL of

Agonist @ EC80

Incubate

90 minutes

@ 37°C*

18

ASSAY PROCEDURE — ALLOSTERIC MODULATOR DOSE RESPONSE The steps outlined below provide the assay volumes and procedure for performing

allosteric modulator assays using PathHunter β-Arrestin GPCR Cell Lines and

PathHunter Detection Reagents in a 384-well format. Refer to Appendix A for cell numbers and volumes for alternate formats. Although plate layouts and experi-mental designs may vary, we recommend performing a 11-point dose curve for each compound using at least duplicate wells for each dilution. The protocol and volumes described below are designed for a complete 384-well plate.

Figure 7. This plate map shows 11-point dose curves with 2 data points at each concentration. Plate map allows 16 modulator compounds to be tested in duplicate per 384-well plate.

DAY 1: PREPARATION OF ASSAY PLATES Plate PathHunter cells in the appropriate number of wells in a 384-well plate as described in the ―Preparation of Assay Plates‖ section on page 9. Allow cells to incubate overnight. DAY 2: MODULATOR COMPOUND PREPARATION AND ADDITION 1. Dissolve your allosteric modulator compound in the vehicle of choice (DMSO,

ethanol, PBS or other) at the desired stock concentration.

2. Prepare a series of eleven 3-fold serial dilutions of modulator compound in CP Reagent containing the appropriate solvent (DMSO, ethanol, PBS or other) as described below. The concentration of each dilution should be prepared at 10X

the final screening concentration (i.e. 2.5 μL modulator compound will be used

in a final volume of 25 μL). For each dilution, the final concentration of solvent

should remain constant. To prepare the 11-point dose curve serial dilutions,

19

we recommend starting with a concentration that is 50X the expected IC50 value for the compound (e.g. 500X IC50 would be the final working concentration).





wells #1-11.

c) Prepare a working concentration of modulator compound in the appropriate AssayComplete CP Reagent.

d) Add 30 µL of the working concentration of modulator compound to well #12.


gently by pipetting up and down. Discard the pipet tip.



g) Repeat this process 7 more times in succession to prepare serial dilutions for the remaining wells, or from right to left across the plate. DO NOT add modulator compound to wells #1 and 2. These samples serve as the no modulator controls and complete the dose curve.

h) Repeat this process for any additional modulator compounds to be tested.

i) Set compounds aside until you are ready to add them to the cells.



on page 17.

5. Incubate cells with modulator compounds for 30 minutes @ 37°C.

AGONIST COMPOUND PREPARATION AND ADDITION 1. During the modulator compound incubation, determine the EC10/EC90 concen-

tration of the agonist from the agonist dose response curve (described on page 10-12). Prepare a 10X EC10 concentration (PAM) or 10X EC90 concentra-tion (NAM) of agonist compound in the appropriate CP Reagent/solvent as shown below:

Example: If the expected EC10/EC90 of the agonist compound is 10 nM, prepare a stock at 100 nM.

2. When the modulator incubation is complete, add 2.5 µL of agonist compound

to well #2-12. Add 2.5 µL of CP Reagent containing appropriate solvent to the

―No modulator/No agonist‖ wells (columns 1 & 13 in Figure 7).


PathHunter -Arrestin GPCR Assays - DiscoverX · 2 contents legal section intended use technology...

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